Immunological and molecular diagnostic assays play a critical role both in the research and clinical fields. Often it is necessary to perform assays for a panel of multiple targets to gain a meaningful or bird's-eye view of results to facilitate research or clinical decision-making. This is particularly true in the era of genomics and proteomics, where an abundance of genetic markers and/or biomarkers are thought to influence or be predictive of particular disease states. In theory, assays of multiple targets can be accomplished by testing each target separately in parallel or sequentially in different reaction vessels (i.e., multiple singleplexing). However, not only are assays adopting a singleplexing strategy often cumbersome, but they also typically required large sample volumes, especially when the targets to be analyzed are large in number.
A multiplex assay simultaneously measures multiple analytes (two or more) in a single assay. Multiplex assays are commonly used in high-throughput screening settings, where many specimens can be analyzed at once. It is the ability to assay many analytes simultaneously and many specimens in parallel that is the hallmark of multiplex assays and is the reason that such assays have become a powerful tool in fields ranging from drug discovery to functional genomics to clinical diagnostics. In contrast to singleplexing, by combining all targets in the same reaction vessel, the assay is much less cumbersome and much easier to perform, since only one reaction vessel is handled per sample. The required test samples can thus be dramatically reduced in volume, which is especially important when samples (e.g., tumor tissues, cerebral spinal fluid, or bone marrow) are difficult and/or invasive to retrieve in large quantities. Equally important is the fact that the reagent cost can be decreased and assay throughput increased drastically.
Many assays of complex macromolecule samples are composed of two steps. In the first step, agents capable of specifically capturing the target macromolecules are attached to a solid phase surface. These immobilized molecules may be used to capture the target macromolecules from a complex sample by various means, such as hybridization (e.g., in DNA, RNA based assays) or antigen-antibody interactions (in immunoassays). In the second step, detection molecules are incubated with and bind to the complex of capture molecule and the target, emitting signals such as fluorescence or other electromagnetic signals. The amount of the target is then quantified by the intensity of those signals.
Multiplex assays may be carried out by utilizing multiple capture agents, each specific for a different target macromolecule. In chip-based array multiplex assays, each type of capture agent is attached to a pre-defined position on the chip. The amount of multiplex targets in a complex sample is determined by measuring the signal of the detection molecule at each position corresponding to a type of capture agent. In suspension array multiplex assays, microparticles or microcarriers are suspended in the assay solution. These microparticles or microcarriers contain an identification element, which may be embedded, printed, or otherwise generated by one or more elements of the microparticle/microcarrier. Each type of capture agent is immobilized to particles with the same ID, and the signals emitted from the detection molecules on the surface of the particles with a particular ID reflect the amount of the corresponding target.
Accurate measurements are highly desirable for analyte detection, e.g., multiplex assays. Microcarrier-based analyte detection assays are often performed using flat microwell assay plates. Such plates allow for high-throughput analysis of multiple samples and/or analytes. However, reading these plates can also introduce systematic errors and/or inefficiencies into the detection process. For example, if the assay plate is not read on a substantially flat detection stage, optical measurements may be inaccurate or variable due to differences in the position of different wells of the plate along the Z-axis. Even if the system is able to account for such differences by changing focus, changing the focus for individual wells wastes time, thereby reducing the efficiency and speed of the assays. Existing methods for measuring flatness of a material are not adapted for calibrating flatness of a detection stage for use in analyte detection on an assay plate (see, e.g., U.S. Pat. Nos. 6,480,802 and 4,538,913; and U.S. PG Pub. No. 2009/0051931).
Therefore, a need exists for methods, systems, devices, and computer-readable storage media that reduce these sources of systematic error and/or inefficiency. Such methods, systems, devices, and computer-readable storage media allow the user to calibrate the assay reading device such that the assay plate is read on a flat detection stage, saving time and resources.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.